Abstract The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [ O-(1-carboxyethylidene)] groups, and half of them were further acylated with d-3-hydroxybutanoyl groups. A much smaller proportion (<5%) of the oligomers was further acylated by a second d-3-hydroxy-butanoyl group. The locations of the substituents were determined chemically and by J-correlated, 1H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.)_ measurements, doubie-resonance 1H-n.m.r. spectroscopy, and 13C-n.m.r. spectroscopy. The composition and structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure ( 1a), the sugar residues are labelled “ a” through “ h”. The main chain ( a–d) is composed of a 4-deoxy-α- l- threo-hex-4-enopyranosyluronic acid group ( a) that is linked to O-4 of a 3- O-acetyl- d-glucosyluronic acid residue ( b) which is β-linked to O-4 of a d-glucosyl residue ( c). Residue c is β-linked to O-4 of the branching d-linked to O-4 of a d-glucosyl residue ( d). The side chain consists of a substituted d-galactosyl group ( h) which is β-linked to O-3 of residue 9 of a β-(1→4)-linked d-glucose trisaccharide (fragment e–f–g). The reducing end of the resulting tetrasaccharide ( e–f–g–h) is β-linked to O-6 of the branching d-glucose residue ( d). In the native polymer, this branching residue is α-linked to O-4 of the modified d-glucuronic acid residue ( a) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated d-glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars ( g and h) of the branch chain bear 4,6- O-(1-carboxyethylidene) groups. The d-galactosyl groups of half of the oligomers are acylated by d-3-hydroxybutanoyl groups at O-3. About 5% of the oligomers bear a second d-3-hydroxybutanoyl group at O-2 of the d-galactosyl group ( h).